First Report of Xanthomonas campestris pv. raphani Causing Leaf Spot Disease of Brassica oleracea in Portugal.

Representing over 25% of the vegetable production, Brassicaceae crops are very important for Portuguese agriculture. Xanthomonas campestris pv. raphani (Xcr) was first described as the causative agent of a leaf spot disease affecting radish and turnip (4). Despite its ability to infect Solanaceae hosts upon inoculation, this pathogen affects mostly Brassicaceae plants. Typical symptoms include circular dark spots that become lighter and are occasionally surrounded by a chlorotic halo. In severely affected leaves, spots were not limited by the veins and coalesced into irregular shapes that perforated the leaves, rendering the plants unsuitable for marketing. In the early 2000s, several isolates causing leaf spots on Brassica oleracea varieties (cauliflower, white cabbage, savoy cabbage, and tronchuda cabbage) were collected in Sintra, near Lisbon. The isolates, identified as putative X. campestris (Xc), formed typical yellow mucoid and convex colonies when grown on YDC medium. Biochemical characterization (2) showed their ability to produce levan and hydrolyze starch and esculin. Isolates were also able to use celobiose, trehalose, glucose, mannose, raffinose, and sucrose. Furthermore, the isolates were oxidase negative and were unable to hydrolyze arginine and to use rhamnose, indol, inositol, and sorbitol, confirming them as Xc. The expected 619-bp amplicon was obtained for all isolates, after PCR using primers DLH120/DLH125 (1). Koch's postulates were fulfilled through pathogenicity tests on B. oleracea cv. Wirosa and Raphanus sativus, hosts susceptible to Xcr as well as to X. campestris pv. campestris (Xcc). Inoculations on B. carinata (Assession No. PI199947) and Solanum lycopersicum, hosts susceptible only to Xcr, were performed to confirm pathovar identification (2). Four plants of each host were inoculated with each isolate by spraying bacterial water suspensions (OD₆₀₀ = 0.1) onto leaf surfaces. Positive and negative controls were performed using Xcc type strain (CFBP 5241) and sterile distilled water, respectively. Plants were kept 15 days with 16-h light and 8-h dark photoperiods at 24 and 18°C, respectively, at RH >80% and checked daily for symptom development. Leaf spots typical of Xcr were observed for all isolates on all hosts 5 days after inoculation. All isolates were recovered after re-isolation from inoculated plants, retaining their initial features. Negative control plants showed no symptoms, while CFBP 5241 caused V-shaped lesions, typical of Xcc, on B. oleracea cv. Wirosa and R. sativus. Further confirmation of the identification was attained by partial sequencing of the gyrB gene, using primers X.gyr.fsp.s1/X.gyr.rsp3 (3). Sequences from four isolates (CPBF 143, Genbank KM094906; CPBF 207, GenBank KM094907; CPBF 209, GenBank GU596416; and CPBF 1171, GenBank GU596419) were compared by nucleotide blast at NCBI and displayed higher levels of DNA similarity (>98%) to NCPPB 1946, the type strain for Xcr, than to NCPPB 528, the type strain for Xcc. The polyphasic approach combining phenotypic and genomic data confirmed the presence of X. campestris pv. raphani in Portugal for the first time. References: (1) T. Berg et al. Plant Pathol. 54: 416, 2005. (2) R. Lelliot and D. Stead. Methods for the Diagnosis of Bacterial Diseases of Plants. Blackwell Scientific Publications, Oxford, England, 1987. (3) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 57:2881, 2007. (4) H. White. Phytopathology 20:653, 1930.